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A team of scientists at Columbia University has created a fully humanized model of amyotrophic lateral sclerosis (ALS) using sporadic ALS astrocytes grown from patients' post-mortem cerebral cortex and/or spinal cord samples and human embryonic stem cell-derived motor neurons.

Carried out in culture, the scientists were able to reproduce the primary features of human ALS; the sporadic ALS astrocytes triggered the selective death of motor neurons. The technique allowed them to identify the death pathway that led to the destruction of the motor neurons. The success of the technique suggests that it could be used to model other human neurodegenerative diseases.

The scientists, led by Serge Przedborski, MD, PhD, the Page and William Black professor of neurology, vice chair for research in neurology, and co-director of Columbia's Motor Neuron Center, reported in the Feb. 6 Neuron that the motor neuron death occurred through necroptosis, a molecularly-regulated form of necrosis.

Since there are currently no definite morphological markers of necroptosis, investigators supported their conclusion by showing that two key proteins in the necroptosis machinery — receptor-interacting protein 1 and mixed lineage kinase domain-like protein — were driving the demise of motor neurons. They are still searching for the toxic factors released by ALS astrocytes that trigger this cascade in motor neuron death.

“This model is derived entirely from human elements,” said Dr. Przedborski. “This is probably the closest, most natural model of human ALS that we can get in a dish.”

Even without knowing the toxin, scientists say that the model that recapitulates ALS in a culture of human cells can be used to identify drugs that target the pathway and prevent motor neuron death. In this study, they were able to block the kinases in this cascade and prevent the death of motor neurons.

Other prior studies, including their own, suggested that there is an unknown toxin involved in familial ALS. In the Neuron paper, the scientists found that the ALS astrocytes were toxic to motor neurons even when mutant superoxide dismutase 1 (SOD1) was not present. “We are trying to track down this toxic factor and learn how it kills motor neurons,” said Dr. Przedborski.

STUDY PROTOCOLS

The investigators removed astrocytes from the motor cortex and spinal cord of six ALS patients who had no evidence of any familial history or mutations associated with the disease. The cells were placed in dishes with healthy motor neurons grown from human embryonic stem cells. A few weeks later, the motor neurons had shrunk and their cell membranes disintegrated. About half of the motor neurons died.

By comparison, no motor neurons died when plated with astrocytes used from autopsied brain tissue from people who did not have ALS. Other cell types tested from ALS patients did not lead to the death of motor neurons. Back in 2007, the same team published a similar finding from astrocytes expressing the mutant form of SOD1.

There is mounting evidence for a toxin, still unknown, that is released from astrocytes and is fatal to motor neurons. When the scientists removed the astrocytes from the media the motor neurons continued to die. For now, they have figured out that the toxin triggers a death cascade in the motor neurons that causes them to explode. If they inhibited one of the two kinases in this pathway, the motor neurons did not undergo cell death.

The obvious putative toxic culprits were SOD1 and TAR DNA-binding protein 43 (TDP43). But reducing levels of the proteins in astrocytes did not stop the death of motor neurons in the presence of the sporadic astrocytes.

It is impossible to know whether scientists can target these kinases in patients and what the effect would be. Would the motor neurons be healthy? The investigators are now doing animal studies to figure it out.

Dr. Przedborski said that this strategy is a way to model human disease in vitro. Transgenic animal model systems are faster and more convenient to use, and they teach scientists a great deal about the disease, but the molecular and pathological findings are not always representative of what takes place in humans, he said, noting that there is also no animal model of sporadic ALS. The human in vitro model is also a way to validate findings from animal studies, he said.

Dr. Przedborski said that the new human cell model of ALS will speed the identification of potential treatments. “We now have access to a simplified model that is relevant to the disease and can therefore potentially be used for high-throughput drug screening. So this model is quite special,” he said.

Dr. Przedborski stressed, however, that the model is preliminary. “We believe if you have something that satisfies the two models [that is, a model made of mouse mutant SOD1 or human sporadic astrocytes] it can lead to a stronger therapeutic candidate than if you just use one model,” said Dr. Przedborski.

TO BE ANSWERED

Questions remain. If there is a toxin produced by astrocytes, why do only half of the motor neurons die? A role for astrocytes in neurodegeneration has been linked to other neurological diseases, specifically Parkinson's, spinocerebellar ataxia type 7, and Rett syndrome.

“If these cell culture findings are faithfully modeling the situation occurring in ALS, then blocking the toxic factor released by astrocytes as early as possible could become an effective neuroprotective strategy against this disease,” Dr. Przedborski said. “Currently, we diagnose ALS at a point when a large number of motor neurons are already gone. As we learn more about astrocytes and the toxic factor or factors they release, we may be able to screen people for elevated levels of these proteins and intervene in a tangible way perhaps even before a person displays any clinical sign of ALS.”

The investigators have been searching for the toxic factor for more than five years.

EXPERTS COMMENT

Creating human cell lines to understand the molecular mechanism and pathology that leads to disease — and using these cell lines to screen for treatments — is becoming a tool of choice for many neurological diseases.

Jeffrey D. Rothstein, MD, PhD, director of the Robert Packard Center for ALS Research at Johns Hopkins University School of Medicine, praised the efforts of the Columbia group to identify the pathway that is involved with motor neuron death, with this caveat: “We must remember that these studies are still in vitro. We have to go back to patients and prove that these events are occurring. Even when we use human cells in vitro, the question remains: Is it also seen in human ALS? And is the pathway drugable?”

Dr. Rothstein added that these types of studies “are a fantastic platform to advance our knowledge of disease.”

Ted Dawson, MD, PhD, the Leonard and Madlyn Abramson professor in neurodegenerative diseases at Hopkins and director of the Morris K. Udall Parkinson's Disease Research Center, agreed. “These techniques can apply to many neurodegenerative conditions. There has been accumulating evidence that astrocytes are secreting something toxic that is leading to the death of motor neurons in ALS. This was a nice confirmation and they were able to reveal the pathways that are involved in this process.”

There have been suggestions that astrocytes may also be producing a toxic substance specific to dopamine cells that are damaged in Parkinson's disease, he said. “If you can identify things that astrocytes are making, then they become potent targets for therapy.”

“This finding in ALS is a beautiful step forward,” said Stanley H. Appel, MD, co-director of the Methodist Neurological Institute. “If we can find a way to inhibit these pathways we could prevent cell death.” He believes that it is important to go back to animal models and replicate the findings, too. “The science we learn from animal models has been phenomenal,” he added.

Junying Yuan, PhD, professor of cell biology at Harvard Medical School, has been at the forefront of research on apoptosis and other roads to cell death. Her group first described necroptosis and identified the proteins involved in triggering this non-caspace death cascade. “The factor that the ALS scientists are looking for is what activates the RIP1 kinase involved in necroptosis,” said Dr. Yuan. “It's interesting but it is still an in vitro study. We need animal model data, and we need to figure out how to properly dose to block this cell death.”

Dr. Przedborski agrees and has been working with Dr. Yuan and her colleagues to do just that. “For the moment, there is no straight forward way to do it,” he said. “We are working to find the proper reagents to validate our findings in patients.”

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